Despite many decades of investigation, the biochemical pathways involved in acute regulation of insulin secretion from islet beta-cells by glucose and other metabolic fuels are incompletely understood. Our group has assembled a set of tools that provide a unique opportunity to gain major new insights in this area, including: 1) Stable cell lines derived from INS-1 insulinoma cells that exhibit either robust or poor glucose-stimulated insulin secretion; 2) A large cadre of recombinant adenoviruses containing genes encoding enzymes or other proteins that alter metabolic pathways in defined ways, thereby allowing testing of key hypotheses: 3) Development of new 13C NMR methods for analyzing metabolic pathways in isolated beta-cells, providing a depth of understanding that is not attainable with more traditional methods. With these tools in hand, we will pursue the following specific aims: 1) To test the long-chain acyl CoA hypothesis of glucose-stimulated insulin secretion, which holds that an intact link between glucose and lipid metabolism is required for glucose sensing; 2) To test the potential role of glutamate and glutamate dehydrogenase in regulation of insulin secretion; 3) To test a new hypothesis concerning the role of pyruvate recycling in regulation of glucose-stimulated insulin secretion. This hypothesis is based on our new finding of increased pyruvate recycling in glucose responsive compare to unresponsive INS-1 derived clones, as evaluated by 13C NMR. It is hoped that the proposed work will eventually guide genetic engineering approaches for creation of cells or cell lines that can be used for transplantation therapy of type 1 diabetes. Knowledge of the basic mechanisms of fuel responsiveness in normal beta-cell preparations is also the necessary prelude for understanding of beta-cell dysfunction in type 2 diabetes and eventual development of improved drug therapies for enhancing insulin secretion.